Bottom Line:
Photobleaching experiments using live cells revealed that LAT-GFP in patches was markedly less mobile than that in nonpatched regions.The decreased mobility in patches was dependent on raft organization supported by membrane cholesterol and signaling molecule binding sites, especially the phospholipase C gamma 1 binding site in the cytoplasmic domain of LAT.Thus, although LAT normally moves rapidly at the plasma membrane, it loses its mobility and becomes stably associated with aggregated rafts to ensure organized and sustained signal transduction required for T cell activation.

ABSTRACTLipid rafts are known to aggregate in response to various stimuli. By way of raft aggregation after stimulation, signaling molecules in rafts accumulate and interact so that the signal received at a given membrane receptor is amplified efficiently from the site of aggregation. To elucidate the process of lipid raft aggregation during T cell activation, we analyzed the dynamic changes of a raft-associated protein, linker for activation of T cells (LAT), on T cell receptor stimulation using LAT fused to GFP (LAT-GFP). When transfectants expressing LAT-GFP were stimulated with anti-CD3-coated beads, LAT-GFP aggregated and formed patches at the area of bead contact. Photobleaching experiments using live cells revealed that LAT-GFP in patches was markedly less mobile than that in nonpatched regions. The decreased mobility in patches was dependent on raft organization supported by membrane cholesterol and signaling molecule binding sites, especially the phospholipase C gamma 1 binding site in the cytoplasmic domain of LAT. Thus, although LAT normally moves rapidly at the plasma membrane, it loses its mobility and becomes stably associated with aggregated rafts to ensure organized and sustained signal transduction required for T cell activation.

fig6: The effect of cholesterol depletion on the mobility of LAT-GFP in patches. (A) Cholesterol in Jurkat cells treated with or without 4 mM MβCD for 40 min was extracted, separated with HPTLC, and visualized with 3% cupric acetate/8% phosphoric acid. (B) Jurkat cells treated with or without MβCD were stimulated with OKT3 for the times indicated. Tyrosine phosphorylation of cellular proteins was analyzed by immunoblotting using anti-PY antibody. (C) Jurkat cells were transiently transfected with an NFAT-luc reporter plasmid, treated with MβCD for 40 min, washed, stimulated with OKT3 or PMA plus ionomycin (P+I) in conditioned culture medium without FCS for 6 h, and then tested for NFAT activity by NFAT reporter assay. (D) After LAT-GFP transfectants were pretreated for 40 min with 4 mM MβCD, the cells were stimulated with poly- l-lysine beads or anti-CD3 beads for 20 min in the presence of 4 mM MβCD. Conjugates were fixed with formaldehyde and observed by confocal microscopy. (E) A selected area (2-μm square) on the LAT-GFP patches in LAT-GFP transfectants treated with or without MβCD was photobleached, and fluorescence recovery was monitored. (F) Bleaching recovery kinetics is represented as the percentage of FRAP for LAT-GFP in patches in the presence or absence of MβCD. Data are representative of three individual experiments.

Mentions:
Next, we wanted to determine the effect of cholesterol depletion on the mobility of LAT-GFP after TCR stimulation. To this end, we used methyl-β-cyclodextrin (MβCD), a reagent that selectively binds and removes cholesterol from the plasma membrane (Klein et al., 1995). Because extraction with 10 mM MβCD for longer than 20 min resulted in significant cell death due to the strong toxicity (unpublished data), we reduced the concentration of MβCD to 4 mM. It was reported that Jurkat cells can be viably cultured for 40 min or more while reducing cholesterol levels to ∼50% with this concentration (Harder and Kuhn, 2000), and we confirmed a similar reduction in cholesterol by lipid analysis using high performance thin layer chromatography (HPTLC; Fig. 6 A). When we analyzed tyrosine phosphorylation of intracellular proteins after TCR stimulation in Jurkat cells treated for 40 min with 4 mM MβCD, we found no significant inhibition in this response (Fig. 6 B). Tyrosine phosphorylation of intracellular proteins and an increase of intracellular Ca2+ (unpublished data) were both clearly induced in response to TCR stimulation in Jurkat cells extracted under this condition, suggesting that extraction with 4 mM MβCD for 40 min does not affect early signaling events after TCR stimulation. However, we observed a clear inhibition of late signaling events with this treatment. Because prolonged treatments with MβCD significantly reduced cell viability even at a concentration of 4 mM, we analyzed the induction of NFAT transcriptional factor in MβCD-treated cells with the following protocol: Jurkat cells were transiently transfected with an NFAT-luc reporter plasmid; treated with MβCD for 40 min; washed; stimulated with OKT3 or PMA plus ionomycin in conditioned culture medium without FCS for 6 h; and tested for NFAT activity. In this assay, we observed a significant reduction in TCR-mediated NFAT activation by MβCD treatment without detriment to cell viability and recovery (Fig. 6 C). Culturing MβCD-treated Jurkat cells in medium containing 10% FCS completely cancelled the inhibition NFAT activity, presumably due to incorporation of exogenous cholesterol in FCS into cells (unpublished data). Together, extraction with 4 mM MβCD for 40 min seemed to impair late signaling events without affecting early signaling events after TCR stimulation.

fig6: The effect of cholesterol depletion on the mobility of LAT-GFP in patches. (A) Cholesterol in Jurkat cells treated with or without 4 mM MβCD for 40 min was extracted, separated with HPTLC, and visualized with 3% cupric acetate/8% phosphoric acid. (B) Jurkat cells treated with or without MβCD were stimulated with OKT3 for the times indicated. Tyrosine phosphorylation of cellular proteins was analyzed by immunoblotting using anti-PY antibody. (C) Jurkat cells were transiently transfected with an NFAT-luc reporter plasmid, treated with MβCD for 40 min, washed, stimulated with OKT3 or PMA plus ionomycin (P+I) in conditioned culture medium without FCS for 6 h, and then tested for NFAT activity by NFAT reporter assay. (D) After LAT-GFP transfectants were pretreated for 40 min with 4 mM MβCD, the cells were stimulated with poly- l-lysine beads or anti-CD3 beads for 20 min in the presence of 4 mM MβCD. Conjugates were fixed with formaldehyde and observed by confocal microscopy. (E) A selected area (2-μm square) on the LAT-GFP patches in LAT-GFP transfectants treated with or without MβCD was photobleached, and fluorescence recovery was monitored. (F) Bleaching recovery kinetics is represented as the percentage of FRAP for LAT-GFP in patches in the presence or absence of MβCD. Data are representative of three individual experiments.

Mentions:
Next, we wanted to determine the effect of cholesterol depletion on the mobility of LAT-GFP after TCR stimulation. To this end, we used methyl-β-cyclodextrin (MβCD), a reagent that selectively binds and removes cholesterol from the plasma membrane (Klein et al., 1995). Because extraction with 10 mM MβCD for longer than 20 min resulted in significant cell death due to the strong toxicity (unpublished data), we reduced the concentration of MβCD to 4 mM. It was reported that Jurkat cells can be viably cultured for 40 min or more while reducing cholesterol levels to ∼50% with this concentration (Harder and Kuhn, 2000), and we confirmed a similar reduction in cholesterol by lipid analysis using high performance thin layer chromatography (HPTLC; Fig. 6 A). When we analyzed tyrosine phosphorylation of intracellular proteins after TCR stimulation in Jurkat cells treated for 40 min with 4 mM MβCD, we found no significant inhibition in this response (Fig. 6 B). Tyrosine phosphorylation of intracellular proteins and an increase of intracellular Ca2+ (unpublished data) were both clearly induced in response to TCR stimulation in Jurkat cells extracted under this condition, suggesting that extraction with 4 mM MβCD for 40 min does not affect early signaling events after TCR stimulation. However, we observed a clear inhibition of late signaling events with this treatment. Because prolonged treatments with MβCD significantly reduced cell viability even at a concentration of 4 mM, we analyzed the induction of NFAT transcriptional factor in MβCD-treated cells with the following protocol: Jurkat cells were transiently transfected with an NFAT-luc reporter plasmid; treated with MβCD for 40 min; washed; stimulated with OKT3 or PMA plus ionomycin in conditioned culture medium without FCS for 6 h; and tested for NFAT activity. In this assay, we observed a significant reduction in TCR-mediated NFAT activation by MβCD treatment without detriment to cell viability and recovery (Fig. 6 C). Culturing MβCD-treated Jurkat cells in medium containing 10% FCS completely cancelled the inhibition NFAT activity, presumably due to incorporation of exogenous cholesterol in FCS into cells (unpublished data). Together, extraction with 4 mM MβCD for 40 min seemed to impair late signaling events without affecting early signaling events after TCR stimulation.

Bottom Line:
Photobleaching experiments using live cells revealed that LAT-GFP in patches was markedly less mobile than that in nonpatched regions.The decreased mobility in patches was dependent on raft organization supported by membrane cholesterol and signaling molecule binding sites, especially the phospholipase C gamma 1 binding site in the cytoplasmic domain of LAT.Thus, although LAT normally moves rapidly at the plasma membrane, it loses its mobility and becomes stably associated with aggregated rafts to ensure organized and sustained signal transduction required for T cell activation.

ABSTRACTLipid rafts are known to aggregate in response to various stimuli. By way of raft aggregation after stimulation, signaling molecules in rafts accumulate and interact so that the signal received at a given membrane receptor is amplified efficiently from the site of aggregation. To elucidate the process of lipid raft aggregation during T cell activation, we analyzed the dynamic changes of a raft-associated protein, linker for activation of T cells (LAT), on T cell receptor stimulation using LAT fused to GFP (LAT-GFP). When transfectants expressing LAT-GFP were stimulated with anti-CD3-coated beads, LAT-GFP aggregated and formed patches at the area of bead contact. Photobleaching experiments using live cells revealed that LAT-GFP in patches was markedly less mobile than that in nonpatched regions. The decreased mobility in patches was dependent on raft organization supported by membrane cholesterol and signaling molecule binding sites, especially the phospholipase C gamma 1 binding site in the cytoplasmic domain of LAT. Thus, although LAT normally moves rapidly at the plasma membrane, it loses its mobility and becomes stably associated with aggregated rafts to ensure organized and sustained signal transduction required for T cell activation.